Activation of the Ras MAP kinase signaling cascade is a central feature common to the majority of human malignancies, making the pathway a potentially high-value target for cancer therapeutics. However, directly targeting canonical elements of this pathway as a therapeutic approach is complicated by systemic toxicities resulting from the fact that Ras MAPK signaling is ubiquitously active in nearly all human tissues, and is required for tissue viability. However, full MAP kinase signaling requires the function of a family of MAPK interacting scaffolding proteins that modulate and orchestrate pathway outputs. Recent data indicates that one of these elements, IQGAP1, which regulates Erk MAPK activity, ?-catenin signaling, cytoskeletal remodeling, and cell motility, is dispensable for normal tissue development and homeostasis, but is required for manifestation of the oncogenic effects of Ras. This proposal aims to define the mechanistic basis for this tumor-specific requirement in a medically-relevant human tissue context. To accomplish this, we will utilize our recently developed three-dimensional human tissue model of genetically-defined invasive epidermal neoplasia generated from primary epidermal keratinocytes expressing tumor-associated active Ras, intact basement membrane, and architecturally faithful native stroma. These models recapitulate hallmark features of epidermal malignancy in vivo including disrupted stratification and differentiation, and invasion through basement membrane into supporting stroma. Engineered tissues will incorporate keratinocytes with RNAi-mediated endogenous IQGAP1 antagonism, combined with expression of IQGAP1 mutants unable to bind specific interacting proteins. These efforts are designed to determine which IQGAP1 functions are most critical for supporting progression to neoplastic invasion. These functional human tissue studies will be important for the design of targeted therapeutics, and are structured to allow for identification of other downstream effectors uniquely required by neoplastic tissue. Second, we will employ additional new genetically-defined human tumor models developed in our laboratory based on expression of different tumor-associated oncogenic driver mutations in primary cells from 12 different tissue types. These engineered neoplasias will be deployed in experiments to define the role of the IQGAP1 scaffold in an array of human tumor types. At the end of the proposed funding period we aim to A) understand the mechanism of IQGAP1 antagonism in epidermal-derived neoplasias as a guide to development of future tumor-selective therapeutics, and B) establish the scope of IQGAP1 necessity in a spectrum of human neoplasias to identify tumor types most likely to be susceptible to inhibition of IQGAP1-mediated processes. PUBLIC HEALTH RELEVANCE: Ninety percent of human malignancies arise at epithelial sites, where progression to malignancy involves the key step of invasion through supporting basement membrane. Because of the technical ability to genetically engineer fully functional 3-dimensional human epidermis both in vitro and in vivo, skin is an ideal epithelial tissue in which to examine this process. The efforts here may lead to identification of new therapeutic targets for prevention and treatment of a range of human cancers driven by common tumor-associated oncogenic drivers.